[Construction of Eukaryotic Expression Vector Containing ROP18-ROP12 of Toxoplasma gondii RH Strain].

OBJECTIVE: To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells. METHODS: Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I . ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene ß-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein. RESULTS: Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind III, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for ß-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2,373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12. CONCLUSIONS: The recombinant eukaryotic plasmid pVAX1-ROP18-ROP-2 is constructed and can be expressed in eukaryotic system.

[Measurement of oxygen concentration using multimode diode laser absorption spectroscopy].

Tunable diode laser absorption spectroscopy (TDLAS) is a widely used technique for high sensitivity, good selectivity and fast response. It is widely used in environment monitoring, industrial process control and biomedical sensing. In order to overcome the drawbacks of TDLAS including high cost, poor stability and center wavelength shift problem. A multi-mode diode laser system based on correlation spectroscopy and wavelength modulation spectroscopy (TMDL-COSPEC-WMS) was used to measure O2 concentration near 760nm at the 1%~30% range of near room temperature. During the experiment, the light is splitter into two beams, respectively through the sample and measuring cell, two receiving optical signal collection containing gas concentration information sent back stage treatment, invert the oxygen concentration through correlation and ratio between measured signal and reference signal, the correlation spectroscopy harmonic detection technique is used to improve the stability of the system and the signal to noise ratio. The result showed that, there was a good linear relationship between the measured oxygen concentration and the actual concentration value. A detection limit of 280 pmm. m in the 1 atmospheric which approved of the same sample. A continuous measurement for oxygen with the standard deviation of 0. 056% in ambient air during approximately 30 minutes confirms the stability and the capability of the system. The design of the system includes soft and hardware can meet the needs of oxygen online monitoring. The experimental device is simple and easy to use, easy to complex environment application.

[Cloning and bioinformatics analysis of rhoptry protein 11 of Toxoplasma gondii].

Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The open reading frame of ROP11 gene was amplified by using a pair of specific primers designed according to the coding sequence of ROP11 gene (Accession No. DQ077905). The RT-PCR product was digested by restriction enzyme EcoR I and Not I, and then ligated into a pGEX-6P-2 vector. The recombinant plasmid was transferred into E. coli XL-Blue. The positive clones was selected by colony PCR, and confirmed by the double restriction enzyme digestion and sequencing. The RT-PCR product was 1,548 bp. The recombinant plasmid was confirmed by colony PCR and double restriction enzyme digestion. Sequencing results showed that the obtained ROP11 gene was 1 548 bp (Accession No. KC456639). There was a high sequence consistency (99%) between the obtained ROP11 gene sequence and the Toxoplasma ROP11 gene from GenBank. Bioinformatics analysis showed that the ROP11 protein (Mr 57,020) consisted of the signal peptide (amino acids 1-26), 12 conservative domains, a serine/threonine protein kinase catalytic domain (amino acids 170-511), and two potential N-glycosylation sites.

[Expression of EGF and SP domain proteins of MASP-2 and preparation of their polyclonal antibodies].

AIM: To clone and prokaryotically express the EGF and SP domain proteins of human mannan-binding lectin associated serine protease-2 (MASP-2), and prepare their polyclonal antibodies and analyze their immunogenicity. METHODS: The cDNA of human MASP-2 was transcripted reversely from total RNA extracted from human fetal liver tissue, and then EGF and SP domain genes of MASP-2 were amplified from the cDNA by PCR. The EGF and SP fragment domain genes were cloned into pGEX-6P-2 expression vector and induced by IPTG to express GST fusion protein. The BALB/c mice were immunized with the purified fusion proteins to generate polyclonal antibodies. The specificity of the polyclonal antibodies was analysed with Western blotting, and the titer was determined by indirect ELISA. RESULTS: We successfully constructed the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP and induced the expressions of the GST-EGF and GST-SP fusion proteins. The specificity of the polyclonal antibodies was higher, and had no cross reaction with other proteins. Indirect ELISA showed that the titer of anti-EGF antibody was more than 1:32 000 and anti-SP antibody was more than 1:40 000. CONCLUSION: The polyclonal antibodies of the EGF and SP domain proteins we successfully obtained were of higher specificity and titer, which provides the basis for further research on MASP-2.

[Cloning, expression and immunoreactivity analysis of rhoptry protein 18 (rop18) from Toxoplasma gondii].

OBJECTIVE: To clone and express rhoptry protein 18 (ROP18) gene of Toxoplasma gondii, and analyze its immunoreactivity. METHODS: The genomic DNA was extracted from T. gondii (RH strain) tachyzoites. TgROP18 gene was amplified by PCR, and cloned into pET30a (+) vector. The constructed pET30a (+)-TgROP18 was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed through SDS-PAGE, and identified by Western blotting with mouse anti-T. gondii serum. RESULTS: The TgROP18 gene was about 1 665 bp in length and encoded for a protein of 544 amino acid residues and the former 47 amino acids consisted signal peptide sequences. PCR, enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET30a (+)-TgROP18 was constructed. Bacteria containing recombinant plasmid pET30a (+)-TgROP18 expressed a soluble protein of His-TgROP18 (M, 59 800) after being induced with IPTG. His-TgROP18 reacted positively with mouse anti-T. gondii serum by Western blotting analysis. CONCLUSION: The soluble His-TgROP18 protein shows certain immunoreactivity.

[Establishment of loop-mediated isothermal amplification (LAMP) for detecting Pneumocystis carinii].

Animal model of Pneumocystis carinii pneumonia (PCP) was established for acquiring lung tissue infected with P. carinii. After DNA from rat lungs was extracted, nuclear ribosome small subunit 18s rDNA of P. carinii was amplified by loop-mediated isothermal amplification method at 63 degrees C for 60 min. The product was digested by restriction enzyme Apal I. The results showed that 18s ribosome DNA (rDNA) of P. carinii was cloned into vector pGEX6p2, and the positive clones screened. Therefore, the loop-mediated isothermal amplification has been established for detecting P. carinii.

[Enzyme change in bronchoalveolar lavage fluid of pneumocystis pneumonia rats and the effect of garlicin treatment].

OBJECTIVE: To study the change of enzymes and effect of garlicin treatment on the change in bronchoalveolar lavage fluid (BALF) of rats with Pneumocystis carinii pneumonia (PCP). METHODS: Wistar rats were injected intramuscularly continually with dexamethasone to establish the rat model of PCP. The experimental rats (group A) were injected intramuscularly with garlicin at a dose of 10 mg/(kg x d) for 5 days in the 3rd, 6th and 9th week respectively, and SMZ/TMP therapy group (B), PCP infected group (C) and normal group (D) were established as controls. Three days after the last treatment, the rats of all groups were killed and BALF was collected without contamination and enzymes AST, ALF, CHE, ALP, LDH, CK, CKMB, HBDH, AFU, 5'NT, ADA were examined. RESULTS: The ALP level in group C [(573.41 +/- 350.63)U/L] was significantly higher than that in group D [(210.56 +/- 114.41) U/L] (q = 4.682, P < 0.01), group A [(392.07 +/- 217.57) U/L] (q = 3.851, P < 0.05), and group B [(325.21 +/- 180.65) U/L] (q = 4.380, P < 0.01); the level of CK, CKMB and 5'NT in group C [948.94 +/- 403.43, 489.47 +/- 254.46 and (6.76 +/- 3.11) U/L respectively] was higher than those in group D [426.22 +/- 319.00, 213.33 +/- 144.54 and (3.22 +/- 1.20) U/L] (q = 4.696, 3.784, 3.812, P< 0.05); there was no significant difference in the level of AST, ALT, CHE, LDH, HBDH, AFU and ADA among the four groups (F = 1.852, 0.958, 2.470, 1.423, 1.178, 1.342, 0.611, P > 0.05). CONCLUSIONS: The level of ALP, CK, CKMB but the ALP level decreases distinctly after the garlicin and 5'NT increases evidently in BALF of PCP infected rats, but the ALP level decreases distinctly after the garlicin treatment.

[Localization of the hypothetical protein CT249 in the Chlamydia trachomatis inclusion membrane].

To characterize the hypothetical protein CT249 using antibodies raised with CT249 fusion protein. The open reading frame (ORF) coding for CT249 in the Chlamydia trachomatis serovar L2 genome was cloned into the pGEX-6p2 vector using the restriction enzymes BamH I and Not I. The recombinant plasmid pGEX-6p2-CT249 was transformed into XL1-blue bacteria and the gene CT249 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CT249 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CT249 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). The CT249 gene with 351bps in length was successfully cloned and expressed as GST fusion protein with molecular weight of 38.2kDa. The anti-fusion protein antibodies produced from mice detected the hypothetical protein CT249 in the inclusion membrane of Chlamydia trachomatis-infected cells. Using antibodies raised with GST-CT249 fusion protein, the hypothetical protein CT249 have been identified as a Chlamydia trachomatis inclusion membrane protein. Given the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells, this finding has provided a useful tool for further understanding the mechanisms of chlamydial intracellular parasitism.

[Correlation between point mutation frequency of MBL gene exon I and plasma MBL concentration in healthy Mongolians].

AIM: To investigate the point mutation at codon 54 of mannose binding lectin (MBL) gene, detect the plasma MBL level, and analyze the correlation between the gene mutation frequency and plasma MBL concentration. METHODS: A method for detecting MBL gene point mutation (PCR-RFLP) was established with self-designed primers according to MBL genomic sequence. The plasma MBL concentration was detected by MBL Oliger ELISA kit. RESULTS: The PCR-RFLP for detecting the point mutation at codon 54 of MBL gene was established. Frequency of point mutation at codon 54 of MBL gene in healthy Mongolians was 0.18. The plasma MBL concentration was (2.53+/-1.96)mg/L. There was negative correlation between plasma MBL concentration and MBL gene mutation frequency in Mongolians (r = -0.641). CONCLUSION: The established method of PCR-RFLP was proved to have high specificity, excellent reproducibility and high sensitivity. The relationship between frequency of mutation at codon 54 of MBL gene and the plasma MBL concentration in healthy Mongolians is negatively correlated.